DESCRIPTION (Verbatim from the applicant's abstract) Sodium (Na) channels, the principle target of local anesthetic agents, change their conformational state in response to membrane potential. Depolarization increases the intensity of the drug-receptor interaction nearly 100-fold. While this phenomenon imderlies the broad therapeutic efficacy of local anesthetics, these voltage-dependent interactions also provoke life-threatening side effects (arrhythmias, seizures). Clarifying this dynamic modulation of the drug-receptor interaction will complement new data identifying local anesthetic binding domains, and will improve the design of these compounds. A number of distinct Na channel loci that mediate intrinsic conformational changes (fast and slow inactivation) also modulate local anesthetic action, consistent with the original proposal (the Modulated Receptor Hypothesis) that local anesthetic binding affinity is governed by conformational state. A corollary prediction is that local anesthetics, when bound, should induce the Na channel to occupy higher-affinity conformational state(s). Recent work reveals that intrinsic Na channel conformational changes involve sizeable molecular motions that modify the architecture of the pore. We propose that local anesthetics function as allosteric effectors to induce intrinsic conformational changes in the pore, analogous to ligand effects on allosteric enzymes. Our studies will examine the gating conformational properties of heterologously-expressed Na channels containing engineered cysteine residues using patch-clamp methods. Using methanethiosulfonate reagents, we will examine how slow inactivation changes the sulfhydryl accessibility of amino acid residues in the outer pore region in response to local anesthetic binding. In addition, we will examine the linkage between the earliest phase of depolarization-induced block and motion of the charged S4 segments, with a view to linking motion of these segments to conformational changes in the outer pore. Finally, we will utilize a disulfide trapping approach to constrain the interdomain distance changes induced by local anesthetic block, and will interpret these using a new molecular model of the Na channel pore. Our studies will illuminate the dynamic structure of the Na channel pore, and will facilitate the design of more targeted and less toxic local anesthetic agents.